Techniques for transcript analysis, such as microarray analysis and serial analysis of gene expression (SAGE™), are indispensable for various types of biological research. Use of a microarray enables the expression analysis of large quantities of genes at one time and simultaneous analysis of multiple samples. With the use of a microarray, however, expression analysis can only be conducted exclusively for the genes spotted on the array. Accordingly, it is necessary to prepare an array upon which all relevant genes may be spotted, in order to perform extensive analysis. In the case of model organisms, such as rice or Arabidopsis thaliana, cDNA arrays or oligonucleotide arrays covering all genes thereof are commercially available and are generally employed in research. Concerning many other organisms, however, researchers are required to independently design arrays from cDNA libraries. This requires large amounts of time and cost.
In contrast, serial analysis of gene expression (SAGE™) enables the search for novel genes and the quantitative expression analysis thereof (Velculescu et al., Science 270: 484-487, 1995). With this technique, the genes are identified based on a 10- or 11-bp sequence located downstream of the restriction enzyme site (CATG), which is located closest to the 3′-end of the transcript, and the expression levels of such genes are analyzed. Accordingly, sequential reading of the sequences located around the 3′-end with the use of a DNA sequencer enables the extensive expression analysis of genes, including unknown genes. However, SAGE™ is not substantially suitable for simultaneous analysis of multiple samples due to the large number of experimental steps required. In addition, 14-bp SAGE™ tags and 21-bp tags that are employed in LongSAGE™ (Saha et al., Nature Biotechnology 20, 508-512, 2002) are too short to assuredly identify genes. Thus, applications of such tags are restricted to model organisms.
In recent years, the present inventors had developed the SuperSAGE system, which is an improvement over SAGE™ (WO 2004/099445; Gene expression analysis of plant host-pathogen interactions by SuperSAGE, Matsumura, H., Reich, S., Ito, A., Saitoh, H., Kamoun, S., Winter, P., Kahl, G., Reuter, M., Krueger, D., and Terauchi R., 2003, Proc. Natl. Acad. Sci. U.S.A., 100: 15718-15723; Molekulares Wechselspiel von Wirt und Pathogen: Simultane, genomweite Transkriptprofilierung zweier Organismen mit SuperSAGE, Kahl, G., Winter, P., Matsumura, H., Reuter, M., Kruger, D. and Terauchi R., 2004, Biospektrum 10: 511-513; SuperSAGE, Matsumura, H., Ito, A., Saitoh, H., Winter, P., Kahl, G., Reuter, M., Krueger, D. H. and Terauchi, R., 2005, Cellular Microbiology, 2005, 7: 11-18; and SuperSAGE, a potent transcriptome tool for eukaryotic Organisms, Matsumura, H., Reich, S., Reuter, M., Krueger, D. H., Winter, P., Kahl, G. and Terauchi R., In: S.-M. WANG (ed.) SAGE: Current Technologies and Applications, Horizon Scientific Press, 2004, 77-90). SuperSAGE involves the use of a type III restriction enzyme, EcoP151, to obtain a 26-bp nucleotide sequence tag. Use of tags each of 26 bp remarkably improves the accuracy of gene identification. Such tags also enable simultaneous analysis of gene expression both in host cells and in pathogen cells, and applications thereof became available with regard to non-model organisms, for which no DNA database is available.